1Department of Neuroscience, Columbia University, New York, NY, USA2New York State Psychiatric Institute, New York, NY, USA3Research Foundation for Mental Hygiene, New York, New York, USA4Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, NY, USA5Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, NY, USA6Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA7Olink Bioscience, Uppsala, Sweden8Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, NY, USA

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

G protein–coupled receptors (GPCRs) play critical roles in cell signaling. Their putative organization as dimers and/or oligomers in living cells has been proposed to fine-tune cell-cell communication by regulating receptor function and pharmacology (1). However, despite extensive evidence supporting the formation of GPCR oligomers in heterologous systems, the existence of such signaling complexes in their native environment remains controversial, mostly because of the lack of appropriate methodology (2). In the absence of a clear demonstration of receptor heteromerization in vivo, it has been difficult to differentiate downstream signaling crosstalk from heteromer-dependent signaling (3). Recently a time-resolved fluorescence resonance energy transfer (FRET) approach (4) was used to demonstrate the existence of oligomers in native tissue, but this strategy requires high levels of native receptor expression and does not yet allow spatial or subcellular resolution. It has been suggested that GPCR heteromer levels can be regulated by drug exposure or by disease state (5), but this too is difficult to assess in vivo, especially in a manner that can be generally applicable to different receptor pairs. Here we have adapted a well-characterized proximity ligation assay (PLA) to confirm the existence of GPCR complexes in striatal brain slices of mice ex vivo.

Material and methods

Animals

All procedures were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Columbia University and the New York State Psychiatric Institute. Wild-type (WT), D2R knockout (KO) (6), and A2AR KO (7) male congenic C57Bl/6j mice (postnatal ages 90–150 days) were used in this study. All animals were group-housed in a standard animal care facility with a 12-h light/dark cycle, in which they had free access to food and water.

Surgeries/viral injections

Viral injections were performed in a biohazard level 2 biochemical cabinet. Animals were anesthetized with a mixture of ketamine and xylazine via intraperitoneal injection and operated on using standard sterile conditions. The skull was drilled to allow the penetration of a fine glass pipet (8–15-µm inner diameter) containing a solution of GFP or D2L-R-mVenus expressing adeno-associated virus (AAV; GeneDetect, Auckland, New Zealand) or myc-CD8a-EGFP expressing lentivirus. The ejection of the virus was performed using a syringe under positive pressure. Each injection in the dorsal striatum consisted of a volume of 1 µL delivered bilaterally by a single injection at the following coordinates relative to Bregma (in mm): antero-posterior 1; ventro-dorsal 3; medial-lateral 1.7. The rate of injection was 0.2–0.3 µL/min. Following the injections, the skull was covered with triple antibiotic ointment, and the wound sutured. Animals were sacrificed 3–8 weeks later for histology and PLA analysis.

Brain tissue preparation

Fixed tissue. For most experiments, mice were anesthetized and perfused intracardiacally with 50 mL ice-cold 4% paraform-aldehyde (PFA) in 0.1 M Na2HPO4/NaH2PO4, pH 7.4, buffer. Brains were postfixed overnight in the same solution of PFA at 4°C. Coronal sections (30 µm) were processed using a vibratome (Leica, Wetzlar, Germany). Slices were collected in a cryoprotective solution (30% glycerol, 30% ethylene glycol in 0.1 M Tris, pH 7.4) and kept at −20°C until processing.

For experiments using fixed frozen tissue, brains were incubated 24 h in 30% sucrose in 0.1 M Na2HPO4/NaH2PO4, pH 7.4, buffer following the postfixation step. Coronal sections (30 µm) were generated using a cryostat (Leica) and then mounted on slides. Slides were kept at −20°C until processing.

Fresh frozen tissue. Mice were anesthetized and decapitated. Brains were quickly removed and frozen in isopentane (−20°C). Coronal sections (30 µm) were generated using a crysotat and then mounted on slides that were kept at −80°C until processing. Note that in the case of fresh frozen tissue, slices were fixed for 5 min in 4% PFA and then rinsed twice in Tris-buffered saline (TBS) before staining.

Immunohistochemistry

Most of the experiments shown in this study were performed on floating sections. For sections mounted on slides, reagents were applied directly on the slides and covered with parafilm to prevent evaporation.